结核分枝杆菌cfDNA在结核病诊断中的发展现状和挑战

doi:10.19982/j.issn.1000-6621.20220228

摘要/Abstract

摘要：

为实现“2035终止结核”的目标,开发新的检测方法迫在眉睫。结核分枝杆菌cfDNA(Mycobacterium tuberculosis cfDNA, MTB-cfDNA)可表明病原体的存在,是结核病(tuberculosis,TB)诊断和治疗监测有吸引力的生物标志物。易于获取的尿液和(或)血液中的MTB-cfDNA可实现对任何年龄组的肺结核和肺外结核的检测。在胸腔积液、腹腔积液和脑脊液等体液样本中检测MTB-cfDNA对TB的诊断性能高于GeneXpert MTB/RIF等基因组DNA检测。但cfDNA作为TB生物标志物的研究仍处于起步阶段,未来需要更大型、纳入患者类型更全面的临床研究来评估其在TB诊断中的效用。随着检测方法的进一步优化,cfDNA检测有可能改善TB诊断并成为“游戏规则的改变者”。

关键词: 分枝杆菌,结核, 基因,细菌, 诊断, 综述文献(主题)

Abstract:

In order to achieve the goal of “End Tuberculosis by 2035”, it is urgent to develop new detection methods. Mycobacterium tuberculosis cfDNA (MTB-cfDNA) can indicate the presence of pathogens and is an attractive biomarker for tuberculosis (TB) diagnosis and treatment monitoring. Easily accessible MTB-cfDNA in urine and/or blood can be used to detect both pulmonary and extrapulmonary TB in any age group. The diagnostic performance of MTB-cfDNA in fluid samples such as pleural effusion, ascites and cerebrospinal fluid is higher than that of genomic DNA assays such as GeneXpert MTB/RIF. Research on cfDNA as a TB biomarker is still in its infancy, larger and clinical studies including more comprehensive patient types are needed to evaluate the utility of cfDNA in TB diagnosis. With further optimization of detection methods, cfDNA testing may improve TB diagnosis and become a “game changer”.

Key words: Mycobacterium tuberculosis, Genes,bacterial, Diagnosis, Review literature as topic

中图分类号:

R378.91+1

彭利君, 方婷婷, 蔡龙. 结核分枝杆菌cfDNA在结核病诊断中的发展现状和挑战[J]. 中国防痨杂志, 2022, 44(10): 1091-1095. doi: 10.19982/j.issn.1000-6621.20220228

Peng Lijun, Fang Tingting, Cai Long. Development and challenges of Mycobacterium tuberculosis cfDNA in the diagnosis of tuberculosis[J]. Chinese Journal of Antituberculosis, 2022, 44(10): 1091-1095. doi: 10.19982/j.issn.1000-6621.20220228

图/表 1

表1

国内外结核分枝杆菌cfDNA诊断结核病的性能评估

第一作者	样本类型	确诊方法	靶标	检测方法	产物大小 (bp)	涂片阳性 (%)	敏感度 (%)	特异度 (%)
Ushio^[28]	血浆	培养	IS6110 gyrB	dPCR	137	100	65(21/33) 29(10/33)	93(18/19) 100(19/19)
Yan $g [29] a$	全血	综合诊断	IS6110	dPCR qPCR	83	-	100(28/28) 50(14/28)	100(28/28) 100(28/28)
Yan $g [29] b$	全血	综合诊断	IS6110	dPCR qPCR	83	-	100(28/28) 54(15/28)	100(28/28) 100(28/28)
Click^[30]	血浆	培养、Xpert	IS6110	qPCR	106	100	45(18/40)	2/3
韩冰^[31]	血浆	涂片、培养	IS6110	qPCR	-	-	79.70(94/118)	95(58/61)
Lyu^[34]	血浆	涂片、培养	IS6110 IS1081	dPCR	71 71	-	40.6(63/155) 27.1(42/155)	93.4(99/106) 93.4(99/106)
Pan^[33]	血浆	培养、病理和分子	IS6110	qPCR	71	45.8	54.2(13/24)	96.5(55/57)
Park^[32]	血浆	培养、分子	IS6110	PCR	-	31.6	80.0(32/40)^c 47.5(19/40)^d	78.1(32/41) 85.4(35/41)
Cannas^[39]	尿液	涂片、培养	IS6110	半巢式PCR	67/129	95	79(34/43)	100(23/23)
Labugger^[43]	尿液	培养	IS6110	PCR	38	60	64(7/11)	8/8
Patel^[42]	尿液	培养	DR区	PCR	38	33	43(75/175)	89(210/237)
Oreskovic^[41]	尿液	Xpert	IS6110	PCR	40	38.8	83.7(41/49)	100(24/24)
Che^[20]	胸腔积液	综合诊断	IS6110	qPCR	-	-	75(45/60)	100(18/18)
寿娟^[21]	胸腔积液	综合诊断	IS6110	qPCR	-	12.0	63(65/103)	100(33/33)
Yang^[22]	胸腔积液	综合诊断	IS6110	qPCR	-	-	69.23(153/221)	100(65/65)
Sharma^[23]	腹腔积液	综合诊断	devR	qPCR	139	-	70.9(22/31)	97.1(33/34)
Haldar^[24]	脑脊液	培养	IS6110 devR	PCR qPCR	200 144	-	85.2(69/81) 87.6(71/81)	92(72/86) 92(79/86)
Haldar^[25]	脑脊液	培养	devR	qPCR	144	-	100(29/29)	96(125/130)
Shao^[26]	脑脊液	综合诊断	IS6110	qPCR	75	-	53.3(32/60)	100(24/24)
Li^[27]	脑脊液	综合诊断	IS6110	qPCR	-	-	56.5(26/46)	100(22/22)

表1

参考文献 47

[1]	张慧, 成君, 屈燕, 等. “三新一加强”结核病综合防治服务模式的验证、示范与推广:中国国家卫生健康委员会-比尔及梅琳达·盖茨基金会结核病项目. 中国防痨杂志, 2021, 43(8):757-760. doi: 10.3969/j.issn.1000-6621.2021.08.001. doi: 10.3969/j.issn.1000-6621.2021.08.001
[2]	Chakaya J, Khan M, Ntoumi F, et al. Global Tuberculosis Report 2020-Reflections on the Global TB burden, treatment and prevention efforts. Int J Infect Dis, 2021, 113 Suppl 1(Suppl 1):S7-S12. doi: 10.1016/j.ijid.2021.02.107. doi: 10.1016/j.ijid.2021.02.107
[3]	Walzl G, McNerney R, du Plessis N, et al. Tuberculosis: advances and challenges in development of new diagnostics and biomarkers. Lancet Infect Dis, 2018, 18(7): e199-e210. doi: 10.1016/S1473-3099(18)30111-7. doi: 10.1016/S1473-3099(18)30111-7
[4]	张少俊, 杨驰, 范琳. 血清结核抗体诊断活动性结核病的价值. 中国防痨杂志, 2018, 40(1): 20-25. doi: 10.3969/j.issn.1000-6621.2018.01.007. doi: 10.3969/j.issn.1000-6621.2018.01.007
[5]	De Groote MA, Sterling DG, Hraha T, et al. Discovery and Validation of a Six-Marker Serum Protein Signature for the Diagnosis of Active Pulmonary Tuberculosis. J Clin Microbiol, 2017, 55(10):3057-3071. doi: 10.1128/JCM.00467-17. doi: 10.1128/JCM.00467-17 pmid: 28794177
[6]	贾红彦, 董静, 张宗德, 等结核分枝杆菌感染的免疫学检测技术研究进展及临床应用现状. 中国防痨杂志, 2022, 44(7): 720-726. doi: 10.19982/j.issn.1000-6621.20220103. doi: 10.19982/j.issn.1000-6621.20220103
[7]	Fernández-Carballo BL, Broger T, Wyss R, et al. Toward the Development of a Circulating Free DNA-Based In Vitro Diagnostic Test for Infectious Diseases: a Review of Evidence for Tuberculosis. J Clin Microbiol, 2019, 57(4):e01234-18. doi: 10.1128/JCM.01234-18. doi: 10.1128/JCM.01234-18
[8]	沙巍. 规范合理使用分子生物学检测技术以早期精准诊断结核病. 中国防痨杂志, 2021, 43(10): 983-986. doi: 10.3969/j.issn.1000-6621.2021.10.001. doi: 10.3969/j.issn.1000-6621.2021.10.001
[9]	Mandel P, Metais P. Nuclear Acids In Human Blood Plasma. C R Seances Soc Biol Fil, 1948, 142(3-4): 241-243.
[10]	Weerakoon KG, McManus DP. Cell-Free DNA as a Diagnostic Tool for Human Parasitic Infections. Trends Parasitol, 2016, 32(5):378-391. doi: 10.1016/j.pt.2016.01.006. doi: S1471-4922(16)00007-6 pmid: 26847654
[11]	Botezatu I, Serdyuk O, Potapova G, et al. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clini-cal chemistry, 2000, 46:1078-1084.
[12]	Reckamp KL, Melnikova VO, Karlovich C, et al. A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma. J Thorac Oncol, 2016, 11(10):1690-1700. doi: 10.1016/j.jtho.2016.05.035. doi: 10.1016/j.jtho.2016.05.035 pmid: 27468937
[13]	Lu T, Li J. Clinical applications of urinary cell-free DNA in cancer: current insights and promising future. Am J Cancer Res, 2017, 7(11):2318-2332. pmid: 29218253
[14]	Gielis EM, Ledeganck KJ, De Winter BY, et al. Cell-Free DNA: An Upcoming Biomarker in Transplantation. Am J Transplant, 2015, 15(10):2541-2551. doi: 10.1111/ajt.13387. doi: 10.1111/ajt.13387 pmid: 26184824
[15]	Tsui NB, Jiang P, Chow KC, et al. High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing. PLoS One, 2012, 7(10):e48319. doi: 10.1371/journal.pone.0048319. doi: 10.1371/journal.pone.0048319 URL
[16]	Lo YM, Chan LY, Chan AT, et al. Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res, 1999, 59(21):5452-5455. pmid: 10554016
[17]	White PL, Barnes RA, Springer J, et al. Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative. J Clin Microbiol, 2015, 53(9):2832-2837. doi: 10.1128/JCM.00905-15. doi: 10.1128/JCM.00905-15 pmid: 26085618
[18]	Waggoner JJ, Balassiano I, Abeynayake J, et al. Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis. PLoS One, 2014, 9(11): e112356. doi: 10.1371/journal.pone.0112356. doi: 10.1371/journal.pone.0112356 URL
[19]	Weerakoon KG, Gordon CA, Williams GM, et al. Droplet Digital PCR Diagnosis of Human Schistosomiasis: Parasite Cell-Free DNA Detection in Diverse Clinical Samples. J Infect Dis, 2017, 216(12):1611-1622. doi: 10.1093/infdis/jix521. doi: 10.1093/infdis/jix521 pmid: 29029307
[20]	Che N, Yang X, Liu Z, et al. Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion. J Clin Microbiol, 2017, 55(5):1526-1532. doi: 10.1128/JCM.02473-16. doi: 10.1128/JCM.02473-16 URL
[21]	寿娟, 谢青梅, 龙昭玲, 等. 胸水游离DNA的结核杆菌检测在结核病诊断中的价值. 中华病理学杂志, 2018, 47(6):465-467. doi: 10.3760/cma.j.issn.0529-5807.2018.06.016. doi: 10.3760/cma.j.issn.0529-5807.2018.06.016
[22]	Yang X, Che N, Duan H, et al. Cell-free Mycobacterium tuberculosis DNA test in pleural effusion for tuberculous pleurisy: a diagnostic accuracy study. Clin Microbiol Infect, 2020, 26(8): 1089.e1-1089.e6. doi: 10.1016/j.cmi.2019.11.026. doi: 10.1016/j.cmi.2019.11.026
[23]	Sharma P, Anthwal D, Kumari P, et al. Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis. PLoS One, 2020, 15(8):e0238119. doi: 10.1371/journal.pone.0238119. doi: 10.1371/journal.pone.0238119 URL
[24]	Haldar S, Sharma N, Gupta VK, et al. Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR. J Med Microbiol, 2009, 58(Pt 5): 616-624. doi: 10.1099/jmm.0.006015-0. doi: 10.1099/jmm.0.006015-0 URL
[25]	Haldar S, Sankhyan N, Sharma N, et al. Detection of Mycobacterium tuberculosis GlcB or HspX Antigens or devR DNA impacts the rapid diagnosis of tuberculous meningitis in children. PLoS One, 2012, 7(9): e44630. doi: 10.1371/journal.pone.0044630. doi: 10.1371/journal.pone.0044630 URL
[26]	Shao L, Qiu C, Zheng L, et al. Comparison of diagnostic accuracy of the GeneXpert Ultra and cell-free nucleic acid assay for tuberculous meningitis: A multicentre prospective study. Int J Infect Dis, 2020, 98:441-446. doi: 10.1016/j.ijid.2020.06.076. doi: S1201-9712(20)30513-0 pmid: 32599283
[27]	Li X, Du W, Wang Y, et al. Rapid Diagnosis of Tuberculosis Meningitis by Detecting Mycobacterium tuberculosis Cell-Free DNA in Cerebrospinal Fluid. Am J Clin Pathol, 2020, 153(1):126-130. doi: 10.1093/ajcp/aqz135. doi: 10.1093/ajcp/aqz135 URL
[28]	Ushio R, Yamamoto M, Nakashima K, et al. Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma. Tuberculosis (Edinb), 2016, 99:47-53. doi: 10.1016/j.tube.2016.04.004. doi: 10.1016/j.tube.2016.04.004 URL
[29]	Yang J, Han X, Liu A, et al. Use of Digital Droplet PCR to Detect Mycobacterium tuberculosis DNA in Whole Blood-Derived DNA Samples from Patients with Pulmonary and Extrapulmonary Tuberculosis. Front Cell Infect Microbiol, 2017, 7:369. doi: 10.3389/fcimb.2017.00369. doi: 10.3389/fcimb.2017.00369 URL
[30]	Click ES, Murithi W, Ouma GS, et al. Detection of Apparent Cell-free M.tuberculosis DNA from Plasma. Sci Rep, 2018, 8(1): 645. doi: 10.1038/s41598-017-17683-6. doi: 10.1038/s41598-017-17683-6 pmid: 29330384
[31]	韩冰, 胡柳杨, 姚亚超, 等血浆结核分枝杆菌游离DNA检测对结核病的诊断价值. 临床肺科杂志, 2020, 25(6):5. doi: 10.3969/j.issn.1009-6663.2020.06.018. doi: 10.3969/j.issn.1009-6663.2020.06.018
[32]	Park JH, Koo B, Kim MJ, et al. Utility of plasma cell-free DNA detection using homobifunctional imidoesters using a microfluidic system for diagnosing active tuberculosis. Infect Dis (Lond), 2022, 54(1):46-52. doi: 10.1080/23744235.2021.1963839. doi: 10.1080/23744235.2021.1963839
[33]	Pan SW, Su WJ, Chan YJ, et al. Mycobacterium tuberculosis-derived circulating cell-free DNA in patients with pulmonary tuberculosis and persons with latent tuberculosis infection. PLoS One, 2021, 16(6): e0253879. doi: 10.1371/journal.pone.0253879. doi: 10.1371/journal.pone.0253879 URL
[34]	Lyu L, Li Z, Pan L, et al. Evaluation of digital PCR assay in detection of M.tuberculosis IS6110 and IS 1081 in tuberculosis patients plasma. BMC Infect Dis, 2020, 20(1):657. doi: 10.1186/s12879-020-05375-y. doi: 10.1186/s12879-020-05375-y
[35]	Green C, Huggett JF, Talbot E, et al. Rapid diagnosis of tuberculosis through the detection of mycobacterial DNA in urine by nucleic acid amplification methods. Lancet Infect Dis, 2009, 9(8): 505-511. doi: 10.1016/s1473-3099(09)70149-5. doi: 10.1016/S1473-3099(09)70149-5 pmid: 19628175
[36]	Elshimali YI, Khaddour H, Sarkissyan M, et al. The clinical utilization of circulating cell free DNA (CCFDNA) in blood of cancer patients. Int J Mol Sci, 2013, 14(9):18925-18958. doi: 10.3390/ijms140918925. doi: 10.3390/ijms140918925 pmid: 24065096
[37]	Oreskovic A, Brault N, Panpradist N, et al. Analytical Comparison of Methods for Extraction of Short Cell-Free DNA from Urine. J Mol Diagn, 2019, 21(6):1067-1078. doi: 10.1016/j.jmoldx.2019.07.002. doi: S1525-1578(19)30354-X pmid: 31442674
[38]	Oreskovic A, Waalkes A, Holmes EA, et al. Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing. Int J Infect Dis, 2021, 112:330-337. doi: 10.1016/j.ijid.2021.09.042. doi: 10.1016/j.ijid.2021.09.042 pmid: 34562627
[39]	Cannas A, Goletti D, Girardi E, et al. Mycobacterium tuberculosis DNA detection in soluble fraction of urine from pulmonary tuberculosis patients. Int J Tuberc Lung Dis, 2008, 12(2):146-151. pmid: 18230246
[40]	Oreskovic A, Lutz BR. Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples.PLoS One 2021, 16(2):e0247851. doi: 10.1371/journal.pone.0247851. doi: 10.1371/journal.pone.0247851
[41]	Oreskovic A, Panpradist N, Marangu D, et al. Diagnosing Pulmonary Tuberculosis by Using Sequence-Specific Purification of Urine Cell-Free DNA. J Clin Microbiol, 2021, 59(8):e0007421. doi: 10.1128/JCM.00074-21. doi: 10.1128/JCM.00074-21
[42]	Patel K, Nagel M, Wesolowski M, et al. Evaluation of a Urine-Based Rapid Molecular Diagnostic Test with Potential to Be Used at Point-of-Care for Pulmonary Tuberculosis: Cape Town Cohort. J Mol Diagn, 2018, 20(2): 215-224. doi: 10.1016/j.jmoldx.2017.11.005. doi: S1525-1578(17)30247-7 pmid: 29269279
[43]	Labugger I, Heyckendorf J, Dees S, et al. Detection of transrenal DNA for the diagnosis of pulmonary tuberculosis and treatment monitoring. Infection, 2017, 45(3):269-276. doi: 10.1007/s15010-016-0955-2. doi: 10.1007/s15010-016-0955-2 pmid: 27798774
[44]	World Health Organization. High priority target product profiles for new tuberculosis diagnostics:report of a consensus meeting. Geneva: World Health Organization, 2014.
[45]	EI Messaoudi S, Rolet F, Mouliere F, et al. Circulating cell free DNA: Preanalytical considerations. Clin Chim Acta, 2013, 424:222-230. doi: 10.1016/j.cca.2013.05.022. doi: 10.1016/j.cca.2013.05.022 pmid: 23727028
[46]	Murugesan K, Hogan CA, Palmer Z, et al. Investigation of Preanalytical Variables Impacting Pathogen Cell-Free DNA in Blood and Urine. J Clin Microbiol, 2019, 57(11):e00782-19. doi: 10.1128/JCM.00782-19. doi: 10.1128/JCM.00782-19
[47]	Burnham P, Dadhania D, Heyang M, et al. Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract. Nat Commun, 2018, 9(1):2412. doi: 10.1038/s41467-018-04745-0. doi: 10.1038/s41467-018-04745-0 pmid: 29925834